Primate embryonic stem cells

Abstract

A purified preparation of primate embryonic stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (-); SSEA-3 (+); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a particularly advantageous embodiment, the cells of the preparation have normal karyotypes and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cell lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed.

We claim: 
     
       1. A purified preparation of primate embryonic stem cells which (i) is capable of proliferation in an in vitro culture for over one year, (ii) maintains a karyotype in which all the chromosomes characteristic of the primate species are present and not noticeably altered through prolonged culture, (iii) maintains the potential to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iv) will not differentiate when cultured on a fibroblast feeder layer. 
     
     
       2. The preparation of claim 1 wherein the stem cells will spontaneously differentiate to trophoblast and produce chorionic gonadotropin when cultured to high density. 
     
     
       3. A purified preparation of primate embryonic stem cells wherein the cells are negative for the SSEA-1 marker, positive for the SSEA-3 marker, positive for the SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, and have karyotypes which includes the presence of all of the chromosomes characteristic of the primate species and in which none of the chromosomes are noticeably altered. 
     
     
       4. The preparation of claim 3 wherein the cells are positive for the TRA-1-60, and TRA-1-81 markers. 
     
     
       5. The preparation of claim 3 wherein the cells continue to proliferate in an undifferentiated state after continuous culture for at least one year. 
     
     
       6. The preparation of claim 3 wherein the cells will differentiate to trophoblast when cultured beyond confluence and will produce chorionic gonadotropin. 
     
     
       7. The preparation of claim 3 wherein the cells remain euploid for more than one year of continuous culture. 
     
     
       8. The preparation of claim 3 wherein the cells differentiate into cells derived from mesoderm, endoderm and ectoderm germ layers when the cells are injected into a SCID mouse. 
     
     
       9. A method of isolating a primate embryonic stem cell line, comprising the steps of: (a) isolating a primate blastocyst;   (b) isolating cells from the inner cell mass of the blastocyst of (a);   (c) plating the inner cell mass cells on embryonic fibroblasts, wherein inner cell mass-derived cells masses are formed;   (d) dissociating the mass into dissociated cells;   (e) replating the dissociated cells on embryonic feeder cells;   (f) selecting colonies with compact morphologies and cells with high nucleus to cytoplasm ratios and prominent nucleoli; and   (g) culturing the cells of the selected colonies.   
     
     
       10. A method as claimed in claim 9 further comprising maintaining the isolated cells on a fibroblast feeder layer to prevent differentiation. 
     
     
       11. A cell line developed by the method of step 9.